Skin lightening products are a fast-growing segment of the beauty industry. In Asia, for many people, having light, porcelain skin is considered the ideal. To meet this demand, Soliance has developed and patented Axolight, an active ingredient with skin lightening properties, made from renewable plant matter.
Through effectiveness tests, Axolight has proved its lightening effect from the first step of human melanosome maturation. By specifically inhibiting the enzyme Tyrosinase Related Protein (TRP-1), which is necessary for the activation of tyrosinase itself, Axolight thereby limits melanogenesis at several levels and significantly decreases skin pigmentation.
Biological context
Skin colour depends on the chemical nature and the quantity of natural melanin pigments present in the upper layers of the skin. There are two types of melanin pigments:
• Eumelanine (brown-black colour).
• Pheomelanin (orange-yellow colour).
These pigments are produced by specialised cells, called melanocytes, in intracellular organelles called melanosomes.
The maturation of melanosomes
Melanosomes have four growth stages:
• During stage I, melanosomes are colourless spherical vesicles.
• During stage II, these vesicles lengthen and form themselves.
• During stage III, the production of melanin begins inside the melanosomes.
• During stage IV, melanogenesis is achieved. Melanosomes containing melanin are moved along arm-like structures before being transferred to keratinocytes.
Enzymes responsible for the melanogenesis
Melanogenesis involves various enzymes that catalyse reactions leading to the formation of melanin pigments. These enzymes have similar structures and common characteristics, however, they are coded by different genes and perform different catalytic activities. The enzymes involved are tyrosinase and the tyrosinase-related proteins 1 and 2, called TRP-1 and TRP-2.
Tyrosinase, TRP-1 and TRP-2
Tyrosinase is the key enzyme in melanogenesis, catalysing the three following reactions:
• The hydroxylation of tyrosine into 3,4-dihydroxy-phenylalanin (L-dopa).
• The oxidation of L-dopa into dopaquinone.
• The oxidation of 5,6-dihydroxyindole (DHI) into indole-5,6-quinone.
TRP-2 transforms dopachrome into 5,6-dihydroxy-indole-2-carboxylic acid (DHICA). Although the role of tyrosinase and TRP-2 in human melanogenesis is clearly defined, the role of TRP-1 remains the subject of numerous studies and debates. Indeed, for a long time we thought that TRP-1 had a DHICA oxidase activity and was involved in the formation of eumelanins. Now it appears that, although this is true for mice, it is not the case in humans.1 But the role of TRP-1 in human melanogenesis is fundamental because people without functional TRP-1 suffer from type 3 oculocutaneous albinism (OCA3).2 Today, the role of TRP-1 is partially explained through its activity on tyrosinase on two levels. Firstly, on tyrosine hydroxylation. This reaction is the limiting stage of melanogenesis because it is a slow reaction. However, TRP-1 has a tyrosinase hydroxylase activity at low levels of tyrosine, which would enable the generation of L-dopa in the premelanosome in order to stimulate the tyrosine hydroxylase activity of tyrosinase in stage III melanosome.3 Secondly, on the stabilisation of tyrosinase. Indeed, recent data shows that TRP-1 forms a complex with tyrosinase, which leads to its activation.4 Besides its role in the biosynthesis of melanin, TRP-1 is also involved in maintaining the melanosome‘s structure and proliferating melanocytes.
Axolight by Soliance Green
Axolight (now referred to as ‘the lightening active’) is a solution rich in arabinoxylooligosaccharides, a product of Soliance Green’s plant fractionation expertise. It consists of chains of xylose linked by ?(1-4) and substituted in O-2 and\or O-3 by arabinose. The average degree of polymerisation of xylose chains is four units and the xylose/arabinose ratio is between 0.2 and 0.3.
Mechanism of action
The lightening active is an innovative plant-based skin-lightening active ingredient, which acts at several levels in melanogenesis, for significant results within 28 days. It acts specifically from the first step, inhibiting the transformation of tyrosine into L-dopa through the inhibition of TRP-1 in the pre-melanosome. Moreover, this inhibition destabilises the TRP-1/ tyrosinase complex, thereby reducing its activity. It also inhibits the transformation of L-dopa into dopaquinone, by directly inhibiting tyrosinase activity.
Inhibition of melanogenesis through tyrosinase (in vitro test)
Protocol
Human melanocytes were incubated for 72 hours at 37°C, in a humid atmosphere with 5% of CO2. Incubation was processed:
• Alone (as control).
• With 0.003% kojic acid (as a positive reference).
• With increasing concentrations of the lightening active.
Melanin measurement
The intracellular melanin content was quantified in the cellular lysates by a spectrophotometric measurement at 405 nm.
Measurement of tyrosinase activity
Tyrosinase activity was quantified in the cellular lysates by spectrophotometric measurement with L-dopa (tyrosinase substrate). After tyrosinase action, L-dopa is converted into dopachrome which is a detectable coloured compound at 490 nm.
Protein measurement
Proteins contained in cellular lysates were quantified using a spectrocolorimetric method (Bradford method). All the results were included in the protein quantity.
Results
The lightening active significantly inhibits (*:<0.05) melanin production as of 0.001%. The effect is dose-dependent. The lightening active significantly inhibits (*:<0.05) tyrosinase activity as of 0.001%. The effect is dose-dependent.
Conclusion
The lightening active significantly decreases melanogenesis, much more effectively than kojic acid at lower concentrations. However, results also show that kojic acid almost completely inhibits tyrosinase, compared to the lightening active. This means that the lightening active is involved in another metabolic pathway of melanogenesis inhibition. It is this effect that will be investigated in the ex vivo test.
Inhibition of melanogenesis through TRP-1 (ex vivo test)
Protocol
Explants from an abdominoplasty (about 10 mm in diameter) on a 33 yearold Caucasian woman were prepared and kept alive in a specific BEM medium (BIO-EC’s explants medium). At D0, D2, D4, D6 and D8, skin explants were applied with 2 mg/cm2 of cream containing 3% of the lightening active. The treated control explants were irradiated every day for 10 days with UVAs (6%-8% UVB). Before irradiations, the culture medium was temporarily replaced with a Hank’s Buffered Saline Solution. After irradiations, the explants were put back in the culture medium. At D10, the following parameters were analysed:
• The explants’ lightness was measured using a Minolta CM 2600 D chromameter.
• The quantity of melanin was visualised on paraffin sections formalin-fixed by silver impregnation, according to the Masson-Fontana method.
• The number of TRP-1 positive melanocytes is measured by immunolabelling on paraffin sections with an anti-TRP-1 monoclonal anti-body (clone Ta99, CA1005, Calbiochem) at 1/25 for two hours at room temperature with a streptavidine biotin amplifier system (Vector, PK-7200) revealed in VIP (SK-4600, Vector). The nuclei were counterstained with Masson hemalun.
Results
Measurement of explant discoloration
Using the lightening active for 10 days, coupled with chronic UV irradiation, induces a lightening of the explant, which corresponds to an increase in lightness (L*) whereas the control explant darkens (decrease of the lightness). The effect is significant (*:p<0.1 vs. control).
Visualisation of melanin and image analysis
Image analysis carried out on 18 microscopic fields for each batch enabled us to discover the surface percentage containing melanin in the basement layer. The results were processed statistically. Applying the lightening active for 10 days, coupled with chronic UV irradiation, induces a significant reduction (*:<0.05) of 18% of the surface containing melanin in the basement layer, compared to the control at D10.
Immunolabelling of TRP-1
The TRP-1 positive melanocytes were calculated lengthways for each explant and the number was extrapolated to the epidermic length in centimeters. Results were processed statistically. Applying the lightening active for 10 days, coupled with chronic UV irradiation, induces a significant reduction (*:<0.05) of 88% of the number of TRP-1 positive melanocytes per centimetre of epidermis compared to the control at D10.
Lightening the skin
Protocol
A panel of 22 middle-aged Asian women (37 years old) applied the following creams on their forearms twice a day for 56 days:
• A cream containing 3% of the lightening active.
• A cream containing 2% arbutin.
Skin coloration was measured with a Colorimeter and a Mexameter.
Results
Variation of the lightness
At 28 days, the cream containing 3% the lightening active had significantly increased the CIE 1976 lightness. The variation observed with the cream containing 2% of arbutin was not significant. At 56 days, the creams containing 3% of the lightening active and 2% arbutin had significantly increased the CIE 1976 lightness. There is no significant difference between the two products.
Variation of the mexametric index
At 28 days, the cream containing 3% of the lightening active had significantly decreased the mexametric index on the test zone compared to the cream with 2% of arbutin. At 56 days, the cream containing 3% of the lightening active had significantly decreased the mexametric index on the test zone. At 56 days, 91% of the volunteers present a lightening effect with the cream containing 3% of the lightening active compared to 82% with the cream containing arbutin.
Conclusion
By using 3% the lightening active, the lightening effect is significant within 28 days. The lightening active presents faster discoloration kinetics than arbutin.
Ecoresponsability
The lightening active is classified as easily biodegradable according to an ultimate biodegradability trial according to the OECD 301F directive (by respecting the window of 10 days after having reached 10% of biodegradation).
Cosmetic uses
Time, sun, pollution and stress all leave visible and irreversible marks on our skin. The lightening active has been created to significantly reduce these effects with three objectives:
• Prevent (e.g. sun protection, age spot prevention care).
• Lighten (e.g. whitening and lightening).
• Prolong (e.g. after-sun).
It is particularly well-suited to skincare and suncare uses for the face, the hands and the body. The recommended dose is between 2% and 3%.
Conclusion
Soliance Green’s expertise in green chemistry, extraction and fractionation of high added value plants has been recognised for several years. Axolight is the new active ingredient patented from this technology approved by ECOCERT. Axolight is a solution rich in oligosaccharides, with a controlled APD of four units, whose skin lightening properties have been demonstrated by numerous biological tests (in vitro, in vivo, ex vivo). The results enable us to assert that Axolight reduces skin pigmentation by specifically targeting tyrosinase and TRP-1 in terms of their action on melanin production.
References
1 Boissy RE, Sakai C, Zhao H, Kobayashi T, Hearing VJ. Human tyrosinase related protein-1 (TRP-1) does not function as a DHICA oxidase activity in contrast to murine TRP-1. Exp Dermatol 1998; 7 (4): 198–204. 2 Sarangarajan R, Boissy RE. Tyrp1 and oculocutaneous albinism type 3. Pigment Cell Res 2001; 14 (6): 437-44. 3 Zhao H, Zhao Y, Nordland JJ, Boissy RE. Human TRP-1 has tyrosine hydroxylase but no dopa oxidase activity. Pigment Cell Res 1994; 7 (3): 131-40. 4 Wu H, Park HY. Protein kinase C-beta-mediated complex formation between tyrosinase and TRP-1. Biochem Biophys Res Commun 2003; 311 (4): 948-53.
